Improving the NADH-cofactor specificity of the highly active AdhZ3 and AdhZ2 from Escherichia coli K-12.
J Biotechnol
; 189: 157-65, 2014 Nov 10.
Article
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| MEDLINE
| ID: mdl-24992211
ABSTRACT
Biocatalysis is a promising tool for the sustainable production of chemicals. When cofactor depending enzymatic reactions are involved the applicability of the right cofactor is a central issue. One important example in this regard is the production of alcohols by nicotinamide cofactor (NAD(P)(+)) depending alcohol dehydrogenases. AdhZ3 from Escherichia coli, which is important for the production of alcohols from biomass, has a preference for NADPH as cofactor. We used a structure guided site-specific random approach, to change the cofactor preference towards NADH and to deduce more general rules for redesigning the cofactor specificity. Transfer of a triplet motif from NADH preferring horse liver ADH to AdhZ3 showed an insufficient switch in the preference towards NADH. A combinatorial site saturation mutagenesis altering three residues at once was applied. Library screening with two different cofactor concentrations (0.1 and 0.3mM) resulted in nine improved variants with AdhZ3-LND having the highest vmax and AdhZ3-CND having the lowest K(m). Asparagine was the most frequent amino acid found in eight of nine triplet motifs. To verify the triplet-motif, two variants of E. coli AdhZ2 DIN and LND were designed and confirmed for improved activity with NADH.
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Texto completo:
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Banco de datos:
MEDLINE
Asunto principal:
Alcohol Deshidrogenasa
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Ingeniería de Proteínas
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Proteínas de Escherichia coli
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Escherichia coli K12
Idioma:
En
Revista:
J Biotechnol
Asunto de la revista:
BIOTECNOLOGIA
Año:
2014
Tipo del documento:
Article
País de afiliación:
Alemania